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Preparation of Single Cell Suspension: A Simple Start with Primary Cell Isolation

   |  January 16, 2023

Primary cells refer to cells that are isolated or harvested directly from living tissue or organs(e.g. human,mouse) by using specific methods and culture in vitro. Primary cells have been isolated in vivo and undergone very few population doublings from the tissue where they are derived, retaining the hereditary features of organism tissues. Therefore, they represent a more representative model of the in vivo state. primary cells could be used in various rearch fields. For example, the effect of drugs/toxins can be investigated on isolated primary cells at the cellular level.

Primary cell isolation uses enzymatic and mechanical methods to digest tissue and dissociate it into the single-cell suspension, which should be cultured in an appropriate environment for later use.
The tissue dissociation protocol needs to be adjusted appropriately for different tissue types from different species.
The general primary cell separation steps are sampling, pretreatment, separation treatment, filtration cleaning, and post-treatment.

Sampling → Pretreatment

Sampling is critical in preparing primary single-cell suspensions, as the quality of samples has a direct effect on the subsequent processing.
In the operation, first, the animals are anesthetized or killed under different experimental requirements, and their corresponding part of the skin is fixed and cut to expose the tissue.

At the same time, there are slight differences in the sampling of different tissue types.

Tissue TypesSampling Precautions
Mucosa Tissue of SkinDelineate the sampling area (generally 2-4cm2).
Visceral TissueTry to avoid vessels when taking samples with full knowledge of tissue distribution to save the trouble of lysing red cells in the subsequent processing.
Tumor TissueWhen recording complete information on tissue condition, select areas with dense tumor cells, avoid necrotic parts and ulceration and peel off irrelevant tissues as much as possible.
Brain TissueSelect the sampling position according to the experimental requirements, and pay attention to the temperature conditions.
Liquid SampleThe sample sources of lymphocytes and blood cells are usually lymphoid tissues or peripheral blood, so it is a must to pay attention to anticoagulation and wash and preserve ascites and other types of cells in time.

Additionally, the following points need to be noted in the operation:

(1) Take strict aseptic manipulation fast.
(2) Control the environmental temperature according to different tissue types.
(3) Always use sharp tools to reduce mechanical damage and operate gently to keep the tissues moist.
(4) Try to remove all the non-related tissues, especially the small residual vessels, etc.
(5) Keep a complete record of information like the tissues’ condition and weight.

(▲Tissue pretreatment)

Isolation processing

There are two general methods used in digestive process: mechanical and enzymatic.

Mechnical digestion breaks down the tissues physically by using a homogenier or cutting, rubbing, and grinding the tissues with scissors, copper mesh, and tissue grinder, respectively. Despite the advantages of low cost, simple, and fast operation, this method causes serious mechanical damage to tissues and produces unsatisfactory cell dispersion effects. Thus, it only works with some soft tissues with a soft texture and relatively loose structure.

Enzymatic digestion refers to use enzyme to deal with samples after pretreatment. With enzyme,the collagen fibers are destroyed and elastic fibers between the tissues while hydrolyzing mucopolysaccharides and other proteins that connect cells. The enzymes can also combine with calcium and magnesium ions in cells to form chelates by chemical substances such as EDTA. The mechanical force generated from such a combination makes cells round and disperses them effectively, especially in epithelial tissues.

When using the enzymatic system, the proper enzyme selection and ratios should be concerned. Trypsin and collagenase are the most often used. Trypsin hydrolyzes proteins. It mainly acts on the peptide bonds linked by lysine or arginine to digest and dissolve the proteins in the intercellular substance to disperse the cells. Therefore, it is applicable to many kinds of tissues.

Collagenase, an enzyme extracted from bacteria, can digest collagen very well. Its strong digestive ability holds for fibrous tissues, epithelial tissues, and cancer tissues, as well as the intercellular substance. Various collagenases are suitable for various tissues.

TypeApplicable Tissue Types
Collagenase IEpithelial, lung, adipose, and adrenal tissues
Collagenase IILiver, bone, thyroid, cardiac, and salivary gland tissues
Collagenase IIIGeneral mammalian tissues
Collagenase IVGeneral mammalian tissues
Collagenase VPancreatic islet tissues and connective tissues

There are also some enzymes with strong dissociation ability but can cause cell damage, such as pronase, and lyases with weak dissociation ability but less damage to cells. It should be adjusted according to the actual sample, or the formula can be adjusted to achieve better results. For example, hyaluronidase and collagenase can be used together to digest intercellular matrix.

Moreover, enzymatic digestion and mechanical shearing can make the tissue evenly dispersed and achieve better processing performance. In addtition, the four important points to be noted are as follows.

(1) Pre-treated tissue masses must be washed in the buffer to avoid affecting subsequent experiments.

(2) As an excessive duration of enzymatic digestion may affect the state of cells, both the quantity and the reaction time of enzymes should be adjusted according to the volume of tissue. During the process, it is necessary to ensure uniform mixing.

(3) Strict control over temperature and humidity must be ensured. The optimum temperature for trypsinization is 56°C, but the cells cannot withstand such a high temperature, so they can only be processed at a maximum temperature of 37°C. In addition, some tissues need to be treated with cold digestion.

(4) Pay attention to the storage conditions of the different components of the kit. The lyophilized powder should be protected from moisture, and repeated freezing and thawing should be avoided after dissolving. For example, since proteolytic enzymes will automatically decompose, it is recommended to dissolve it immediately before use and store it on the ice at 2°C to 8°C.

Usually, the preparation process of primary cells is done manually, especially mechanical digestion is difficult to ensure the repeatability of the experiment. RWD single cell suspension dissociator uses tissue processing tubes, tissue dissociation kits, and built-in optimization programs for preparing high-activity single-cell suspension and tissue homogenate.

The preparation of single-cell suspension with high cell viability can be completed within 15-30 minutes, which makes the experiments more reproducible. 4 or 8 independent operating channels and heating jackets can automate the processes and improve the efficiency of tissue processing. It is widely used in single-cell sequencing, primary cell culture, fluorescence activated cell sorting, cell therapy, and other research fields.

Filtration and washing → Posttreatment

Certain procedures need to be done at the end of the single-cell suspension preparation process, including the filtration, centrifugation, and medium replacement, the cell counting and viability analysis of the single-cell suspension. Then, harvest the cell suspension for other downstream applications.

(▲Counting live cells)

Primary cell counts can be performed more accurately using an automated cell counter. RWD automated cell counter is equipped with multiple fluorescence channels, captures bright field and fluorescence images to calculate the concentration of cells in suspension, and clearly presents the counting results and cell morphology. It is suitable for cell analysis in research fields such as immunology and vaccine development, cell therapy, tumor research, stem cell, and metabolism research.

In the last step in the process of primary cell isolation, incubators are frequently used to provide stable cell culture conditions. The RWD D180 CO2 incubator provides a more suitable culture environment for cells by controlling temperature, CO2 concentration precisely and by maintaining a high relative humidity (rH). The high-efficiency (HEPA) filter combined with the 140°C high-temperature dry-heat sterilization method enables to filter particles in the air and reach ISO 5 air cleanliness. It is an indispensable cell culture tool in the fields of stem cell research, immunology research, neurobiology research and cancer research.

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